Elevated PRP19 expression targets Bcl-2 and CASP3 expression to inhibit human lung adenocarcinoma cells apoptosis in vitro

Aims: The gene product of PRP19 besides constituting a central player in the spliceosomal machinery, functions in DNA damage repair, a process that aborts the hasty and unwarranted destruction of cell harbouring irreparable DNA damages by apoptosis. Such DNA alterations are common features underlying tumorigenesis. Based on our earlier report that PRP19 overexpression is inhibitory to proliferation in lung tumor cells, we further investigated the effect of elevated expression of PRP19 on cell survival. Methods: PRP19 expression was augmented in cultured A549 cells via plasmid transfection. Growing cells were subjected to various cell survival and apoptosis assays including CCK-8, DAPI, TUNEL and FITCAnnexin V staining. Lysates were obtained from harvested cells for immunoblotting for the assessment of expression of key apoptotic proteins. Results: Lung adenocarcinoma Benjamin Arko-Boham1, Isaac Okai2, Shujuan Shao3 Affiliation: 1Department of Medical Laboratory Sciences, School of Biomedical and Allied Health Sciences, University of Ghana, Accra, Ghana; 2Department of Human Anatomy, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana; 3Liaoning Provincial Key Laboratory for Proteomics, Department of Histology and Embryology, Dalian Medical University, 9 Western Section, Lvshun South Road, Dalian, China, 116044. Corresponding Author: Benjamin Arko-Boham, P.O. Box KB 876, Korle-Bu, Accra, Ghana; Email: barko-boham@ ug.edu.gh Received: 13 April 2016 Accepted: 28 June 2016 Published: 07 July 2016 cells A549, overexpressing PRP19 via plasmid transfection exhibited delayed onset of apoptosis thereby prolonging their life span. Further test by western blot on key proteins involved in apoptosis regulation revealed that PRP19 overexpression led to augmented expression of anti-apoptotic Bcl-2 proteins while diminishing the expression of caspase-3. The expression of pro-apoptotic protein Bax, was unaltered among test and control groups. The Bcl-2 higher expression coupled with suppression of caspase-3 possibly underlies the in vitro inhibition of apoptosis following PRP19 upregulation. Conclusion: PRP19 overexpression resulted in a modest suppression of apoptosis to prevent the hasty destruction of cells with compromised genomic integrity. This is beneficial to the cell and may explain why PRP19 expression in tumor tissues is higher than in non-tumor tissues.


INTRODUCTION
There are various forms of cancer with respect to the organs or tissues affected and have become a major clinical force battling humanity. Globally, the most common cancers diagnosed annually are those of the lungs, prostate, breast and cervix [1,2]. Lung cancer accounts for the highest number of all annual global cancer-related deaths and remains the leading cause of death of all global cancer related mortalities. Lung cancer was responsible for approximately 1.8 million (12.9% of the total) new cancer cases and 1.6 million cancer-related deaths (19.4%) worldwide in the year 2012 alone [3][4][5]. This, undoubtedly, makes the disease a frightening case that needs more attention. The disease is characterized by uncontrolled cell growth within lung tissues. If untreated, cells may grow and spread to other parts of the body apart from the lungs which usually leads to death. In spite of improvements in diagnosis and treatment, a 5-year survival rate remains at an abysmal 13-15% [6,7]. Adenocarcinoma, the most frequently diagnosed histological subtype accounts for about 40% of all lung cancer cases [3,6,[8][9][10][11]. Biomarker identification for clinical use has been a major focus for biomedical research in the past decades. Although there has been significant advancement, a key setback to the discovery of new cancer biomarkers for effective clinical utilization in diagnosis and therapy remains the relatively nonspecific nature of prospective biomarker candidates. This notwithstanding, several biomarkers for some cancers including lung cancer have been discovered and are currently clinically available [12,13]. In view of this, there is need for more dedicated research aimed at discovering novel dependable and efficient cancer biomarkers for clinical utilization giving this work the needed impetuous.
We have earlier reported on the anti-proliferative effects of PRP19 overexpression in tumor cells [14] and now proceed to provide further evidence on repressive ability of the gene's amplification on apoptosis in lung tumor cells. The gene, with other names such as Pso4, PRP19 and NMP200 has the chromosomal location 11q12.2 and encodes for a 55-kDa protein with both cytoplasmic and nuclear presence [15][16][17][18]. The structure of the gene-product, according to Mahajan and Mitchell [16] is composed of an N-terminus, a C-terminus and an intervening homology domain. The N-terminus has a U-box with E3 ligase activity while the C-terminus is a tryptophan-aspartate 40 (WD-40) repeat domain for support and scaffolding [19]. Reports suggest the gene and its product to function in mRNA splicing, DNA damage repair, ubiquitination, oxidative stress response, transcription elongation and as a structural component of the nuclear membrane [17,[20][21][22][23][24][25].
Quantitative analyses of protein expression levels, as supported by a growing body of evidence, reveal that PRP19 is overexpressed in tissue samples of many human tumors compared to their non-tumor counterparts [17,26]. It, however, remains unclear the underlying reason for the upregulation in the gene's expression in situ during tumorigenesis; as to whether it is of a direct and holistic beneficial value to the cells or a mere stress response mechanism that prompts the initiation of other vital reactions towards survival similar to that of p53 in response to genomic insults. Indeed, Dellago et al. [25] reported that PRP19 expression is increased in different cell types in response to oxidative stress. In vitro experimental evidences seem to suggest that PRP19 upregulation is profitable to cells owing to its known pro-survival properties. These pro-survival activities hinge on PRP19's DNA damage repair ability. Several reports indicate that upregulated PRP19 expression in vitro led to increased resistance to apoptosis and p53 phosphorylation, whereas cells with depleted PRP19 showed significantly diminished resistance to apoptosis which inadvertently resulted in truncated cellular life span [17]. It is therefore worthwhile, investigating the relevance of the observed alteration in the gene's expression in other cancers such as lung cancer in the attempt to decipher mechanisms underlying tumor development and progression which is vital to finding new cancer biomarkers.

Selection of Cell line for in vitro Modeling
A range of lung cancer cell lines and one cervical cancer cell line were screened for PRP19 expression by western blotting. Whole cell lysates were extracted from cultured cells for western blotting. Since overexpression of the gene was intended, the cell line with the lowest PRP19 expression was selected as most suitable candidate for in vitro modeling. Cells used in the study were revived cells which had been stored (5-9 passages) in liquid nitrogen for four to six months. The cells had previously been received from the National Institute of Biologic Sciences (Beijing, China). The cell lines used were SPCA, A549, H446, H460, H520, 95C, 95D, HBE and Hela cells.

Cell Culture and Transfection Conditions
Cells were cultured in RPMI1640 culture medium (Gibco Life Technologies, China) supplemented with 10% fetal bovine serum (FBS) (Hyclone Thermo Scientific, China) without antibiotics and grown in a humidified incubator at 37 o C and 5% CO 2 atmosphere. At between 80-90% confluence, cultures were detached using 0.1% trypsin and 0.02% EDTA for subculturing. For transfection, 6x10 3 cells per well were plated in 24well plates and allowed to grow to 60-70% confluence. A549-pEX-PRP19 and pEX-A549 cells were established by transfecting A549 cells with a pEX vector construct (Suzhou GenePharma, China) containing all regulatory elements using lipofectamine 2000 transfection reagent (Invitrogen, USA) in accordance with manufacturer's instructions. Transfected cells were stably selected with 150 µg/ml geneticin (G418), (Gibco Life Technologies, Thailand) 24 hours post-transfection.

Whole Cell Lysates and Immunoblotting
Growing cells were harvested by trypsinization and pelleted by centrifugation and suspended in appropriate volumes of RIPA buffer (Beyotime, China) with 1% PSMF for protein extraction as directed by manufacturer. Between 150 and 300 µg of protein was separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions, blotted onto nitrocellulose membrane and blocked in 5% non-fat milk in 1X PBS for 1 hour. The membrane was incubated with the primary antibody at the appropriate dilution overnight at 4°C. Primary antibodies for PRP19 (Epitomics, USA) and all other proteins were diluted at 1:800 and 1:100 respectively. The antibodies used included polyclonal anti-caspase-3 (Abcam, UK), polyclonal anti-Bax (Proteintech, USA), polyclonal anti-NF-κB (Proteintech, USA), monoclonal anti-β-actin (Proteintech, USA), monoclonal anti-Bcl-2 (Thermoscientific, USA) and monoclonal anti-RB (Epitomics, USA). The secondary antibodies [goat-anti mouse and goat-anti rabbit (Thermoscientific, USA)] used were HRP coupled, diluted at 1:1000 and detected by chemiluminescence using ChemiDox™XRS+. β-actin was used as internal standard and normalizer.

4', 6-Diamidino-2-Phenylindole (DAPI) Staining
Growing cells were trypsinized and transferred into 96-well plates at a seeding density of 10 4 cells per well. Cell death was induced 24 hours post seeding with 30 µM cisplatin (Hansoh Pharmaceuticals Limited, China) for 0,24 and 48 hours for the different cell groups. Control groups received no cisplatin treatment. After the incubation period, cells were washed with 1 µg/ml DAPI (Roche, Germany) in methanol and then stained with the same concentration of DAPI-methanol solution for 15 minutes at 25 o C in the dark after which cells were viewed under fluorescent microscope. Apoptosis-induced nuclear and cell membrane morphological changes were used to determine apoptosis. To estimate overall level of apoptosis within groups, cells in 10 randomly selected microscope fields (at x40) were counted. To calculate percentage apoptosis the ratio of apoptotic cells to total number of cells was multiplied by 100.

Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay
Growing cells were trypsinized, detached and transferred into 96-well plates at a seeding density of 1.5x10 4 cells per well. Cell death was induced 12 hours post seeding with 30 µM cisplatin for 0, 24 and 48 hours for the different cell groups. Control groups received no cisplatin treatment. After cisplatin incubation period, cells were washed with 1X PBS and then covered with fixation solution (4% paraformaldehyde in PBS) for 1 hour at room temperature. Cells were rinsed with 1X PBS and subsequently covered with blocking solution (3% H 2 O 2 in methanol) for 10 minutes at room temperature. Cells were again rinsed with 1X PBS and treated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 5 minutes on ice. Cells were rinsed with 1X PBS, covered with 50 µl of TUNEL reaction mixture (Roche, Germany) and incubated for 1 hour at 37 o C in darkness in a humidified atmosphere. Cells were finally rinsed 3 times with 1X PBS and visualized in a drop of 1X PBS under fluorescent microscope. TUNEL positive cells in 10 randomly selected microscope fields (at x40) were counted and their mean computed.

Fluorescein Isothiocyanate (FITC)-Annexin V Staining
Growing cells were treated with trypsin (without EDTA) and detached. Cells were washed with 1X PBS and collected by centrifugation at 2000 rpm for 5 minutes. The supernatant was removed and the pelleted cells resuspended in 500 µl of Annexin V binding buffer (Roche, Germany). Equal volumes (5 µl) of FITC solution and Propidium Iodide (PI) solution (Roche, Germany) were added to each cell suspension and incubated for 5 minutes at room temperature. Cells were then visualized and analyzed by FACS (BD Bioscience).

Stress Response Studies
Normal untransfected A549 cells growing under optimum conditions were introduced to different stressinducing agents including; chemical (Geneticin) (Gibco, China), starvation and elevated temperature (heat shock at 42 o C) for 2-3 hours. To serum starve the cells, healthily growing cells were incubated under optimum culture conditions for three hours with culture medium without FBS. For heat shock, healthily growing cells under optimum culture conditions were transferred into an incubator preheated to 42 o C and incubated for 2 hours. In the chemical stress induction, growing cells were treated with growth medium (with FBS) containing 100 ug/ml geneticin (G418), (Gibco Life Technologies, Thailand) and incubated at optimum culture conditions for three hours. Cells were subsequently harvest and suspended in lysis buffer to obtain whole cell lysates for western blotting.

Statistical Analysis
The results presented are the mean and standard deviation of three independent experiments. Students' unpaired t-test was performed to compare groups using

A549 cells have lowest PRP19 expression among selected cell lines
To select an appropriate cell line for in vitro modeling, a range of lung cancer cell lines were screened for PRP19 expression. A549 cell line had the lowest PRP19 expression among the cell lines ( Figure 1A) and was therefore selected as ideal candidate cell line for in vitro modeling since overexpression of the gene was intended. PRP19 expression in A549 cells was successfully augmented ( Figure 1B-C) via plasmid transection

PRP19 overexpression impedes apoptosis
Results from all apoptotic assays suggested that PRP19 upregulation delayed the onset of apoptosis and significantly retarded its progress. In a DAPI experiment, which estimates the level of apoptosis by the degree of nuclear condensation or fragmentation, A549-pEX-PRP19 test group showed a lower rate of apoptosis compared to control groups at all experimental time points of 0, 24 and 48 hours (Table 1) (Figure 2A-B). By 48 hours, an average of 76.6 ± 2.8% of cells of the control groups was in various stages of apoptosis with the predominant being Stage 2 and Stage 3 (nuclear collapse/disassembly stage) of apoptosis. The test group, however, registered 59.3 ± 2.1% of cell being apoptotic with a balanced distribution among Stage 1 and Stage 2 of apoptosis and a minimal number in Stage 3 of apoptosis (Figure 2A-B).
Similarly, a TUNEL assay produced results that conformed in trend with that of the DAPI experiment. A549-pEX-PRP19 cell group had reduced numbers of TUNEL positive (apoptotic) cells compared with control groups at various experimental time points (Figure 3  A-B). At the end of 48 hours of cisplatin treatment, control groups had an average of 84 ± 11 (SD) TUNEL positive cells per microscope field (taken at X40) as against that of 45 ± 8 (SD) TUNEL positive cells per microscope field for the test groups taken at the same magnification (Table 2) with p-value of <<0.001.

PRP19 Overexpression delays onset of Apoptosis
Subjected to FITC-Annexin V experimentation, it was evident that A549-pEX-PRP19 cells showed a delayed onset of apoptosis compared to cells of control groups. Following 24 hours of cisplatin treatment, just about 3% of the total cell population of the test group was found in the early stage of apoptosis compared to the over 8% of the cell population of control groups (Figure 4). After 48 hours of cisplatin treatment, total percentage apoptosis (early stage and late stage apoptosis) for the A549-pEX-PRP19, A549-pEX and A549 experimental groups were 11.62 ± 1.20%, 14.44 ± 0.94% and 19.43 ± 1.10%, respectively with a p-value of 0.035 (Table 3).

PRP19 overexpression amplifies Bcl-2 expression but diminishes CASP3 expression
A further test on some important proteins associated with and involved in apoptosis produced the following observation shown in Figure 5. Whereas the antiapoptotic Bcl-2 showed elevated expression in A549-pEX-PRP19 cells compared to cells of control groups, the pro-apoptotic Bax had uniform expression among both test and control groups. The potent effecter protein of apoptosis, caspase-3 on the other hand, showed diminishing abundance in test groups cells compared with cells of control groups.

PRP19 expression is augmented under stress conditions
When A549 cells were subjected to various stress inducing conditions, augmentation of PRP19 expression was revealed by western blotting. Whole cell lysates from A549 cell groups subjected to 2 to 4 hours of starvation, heat shock at 42 o C and geneticin treatment all showed comparable elevated levels of PRP19 expression ( Figure  6A). Consequently, certain proteins that are known to be involved in the stress response pathway and cell cycle control were assayed for by western blotting. The results ( Figure 6B) showed elevated expression of both retinoblastoma protein (RB) and NF-κB in test group cells compared to cells of control groups.

DISCUSSION
A pro-survival advantage PRP19 upregulation conferred on cells was delayed onset of apoptosis. In all apoptosis related assays conducted in this study, PRP19overexpressing cells exhibited lowered rates of apoptosis compared to cells of control groups. In the DAPI experiment (Table 1 and       at 48 hours of cytotoxic treatment had the predominant apoptotic cells at the second stage with a relatively low number at apoptosis stage 3. At hour 24 of treatment, only 32% of the cells showed visible signs of apoptosis and were largely of the stage 1 (the nuclear condensation stage). Microscopically, visible intracellular and nuclear morphology has been used to delineate apoptosis progression into three overlapping stages; Stage 1 (ring stage), Stage 2 (necklace stage) and Stage 3 (nuclear collapse/disassembly) [27]. In the test groups, entry Edorium J Tumor Bio 2016;3:9-18.

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Arko-Boham et al. 14 Figure 5: Western blotting results for some key proteins involved in apoptosis. CASP3 expression was lower in test groups than in control groups. Bcl-2 was highly expressed in test group cells than those of control groups whereas expression of Bax was unaffected among cells of both test and control groups. β-Actin was used as internal standard and normalizer.   Figure 3) indicating upregulated PRP19's potency at counteracting the cytotoxic effects of cisplatin which lead to cell death by apoptosis. Results from the FITC-Annexin V experiments also supported the point that PRP19 upregulation delayed the onset of apoptosis when fewer cells of the test group were in early stage of apoptosis compared to control groups which had comparatively higher numbers (Table 3 and Figure 4) at the same experimental time points.
In a bid to gain more insight into the mechanism that underlies the above observation, the expression of certain proteins that are important players in apoptosis were assessed. Bax (Bcl-2-associated X protein), showed uniform expression among all groups ( Figure 5) while the expression of Bcl-2 (B cell lymphoma 2) was elevated in cells of test group than those of control groups. Also, Caspase-3 expression was downregulated in cells of the test group than in their counterparts from the control groups. These findings were not unexpected looking at the functions of each of these proteins. Bax is a member of pro-apoptotic family of proteins which induce the opening of the mitochondrial voltage-dependent anion channels resulting in the release of cytochrome-c and other proapoptotic factors from the mitochondria, eventually leading to activation of caspases [28,29]. They function as protagonist proteins of apoptosis to ensure that cells harboring unrepaired damages are quickly destroyed and eliminated. The uniform Bax expression among both test and control groups indicates that cells of all the groups www.edoriumjournaloftumorbiology.com Arko-Boham et al. 15 were receiving equivalent degrees of cellular and genomic insults induced by cisplatin treatment. The difference, however, was the upregulated expression of Bcl-2 in the test group relative to controls. Bcl-2 is a member of the anti-apoptotic family of proteins which function to antagonize apoptosis. Bcl-2 in particular, functions specifically to inhibit the release of cytochrome-c, an important molecule for apoptosis initiation as part of the intrinsic apoptosis pathway [30][31][32][33].
Again, caspase-3 was of interest and the trend observed in its expression with PRP19 overexpression was intriguing. Caspase-3 is a major executioner protein (effector caspase) of apoptosis which is activated in apoptotic cells by both extrinsic (death ligand) and intrinsic (mitochondrial) pathways. It is present in cells as a zymogen and remains dormant until it is cleaved to activation by initiator caspases such as caspases 8 and 9 following initiation of apoptotic signaling events [34,35]. Its expression positively associated with the degree of apoptosis among experimental groups. Control groups which had greater percentages of apoptosis equally had higher expression of caspase-3 as against the test group in which there was reduced rate of apoptosis. In brief, PRP19 overexpression led to augmented expression of anti-apoptotic proteins especially, Bcl-2 and diminished expression of caspase-3, the apoptosis effector enzyme. This ultimately resulted in a slowdown of the rate of apoptosis.
On stress response, the increase in PRP19 expression in response to different forms of stress suggests that the gene may be among the early acting genes in stress response. In stress response, the cell adopts multiple strategies to battle for survival against the offending agent. These strategies include cell cycle arrest that allows time for DNA damage repair in the cells. The observed elevated expression of retinoblastoma (RB) under stressing conditions ( Figure 6B) therefore buttresses our earlier report where PRP19 overexpression promoted cell cycle arrest [14]. Members of the RB protein family function as molecular switch controlling G1 to S-phase transition serving as either transcription repressors or activators depending on their phosphorylation state [36]. When RB is hypophosphorylated it binds to E2F family of transcription factors and works as a transcriptional repressor. When adequately phosphorylated (by cyclin D and E), E2F dissociates from RB allowing it to transcribe genes necessary for DNA syntheses during the S-phase [37,38].
Also, NF-κB is a protein complex that regulates DNA transcription, cytokine production and cell survival through its important role in cellular responses to stimuli including stresses [39]. In its inactive form, NF-κB has cytoplasmic location complexed with its repressor IκBα. When activated NF-κB is translocated into the nucleus where it binds to specific sequences of DNA response elements to set in motion a cascade of events that lead to transcription of downstream DNA and ultimately results in alteration of cell function [39][40][41]. The elevated NF-κB expression as observed in our work ( Figure 6B) under stressing conditions therefore suggests attempts by the cells to survive the deleterious effects of the stress. Such survival attempts were enhanced by PRP19 overexpression since PRP19 overexpressing cell group had higher NF-κB expression under stress compared to control groups. More work however, needs to be done to elucidate the seeming PRP19-NF-κB pathway that is played out during cellular responses to stresses.
In summary, PRP19 overexpression conferred prosurvival advantages on cells which may underpin its upregulated expression in lung tumor tissues and other human tumors. PRP19 overexpression enhances cell survival by delaying the onset of apoptosis and retarded it progress. This is by means of PRP19's influence on some proteins involves in apoptosis. In particular, expression of anti-apoptotic genes is enhanced while that of apoptotic effector enzymes is inhibited. This inadvertently prevents the premature destruction of cells harbouring DNA damages in the bit to possibly achieve repair of the damages.

CONCLUSION
Overexpression of PRP19 resulted in a modest suppression of apoptosis to prevent the hasty destruction and removal of cells with compromised genomic integrity. The gene's expression in cells is upregulated in conditions that induce DNA damages. This may explain why its expression in tumor tissues is higher than in non-tumor tissues, an indication of accumulation of DNA damage which is quintessential of many cancers. In the light of intense search for cancer biomarkers for